Anti-viral agents prepared from sea lettuce as raw material

ABSTRACT

Antiviral agents containing sea lettuce or an organic solvent extraction fraction thereof as an active component, and feeds containing the antiviral agents are disclosed. In addition, a process for preparing the antiviral agents is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based upon and claims the benefit of priorityfrom the prior Japanese Patent Application No. 2001-104860, filed Apr.3, 2001, the entire contents of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to use of sea lettuce, a kind ofseaweed, and its extract as feed and medicines. In particular, theinvention relates to use of the antiviral component contained in the sealettuce.

[0004] 2. Description of the Related Art

[0005] In fish-farming, there has often seen the overcrowded cultivationthat attached great importance to productive efficiency, and because ofsuch the cultivation, various fish diseases to induce death of thecultivated fish have been reported. Among such fish diseases, variousmedicines such as antibiotics can control fish diseases in the event ofonset caused by bacteria and so on, and the control of fish diseases byuse of antibiotics has already matured into a particular industrialfield in cultivation business.

[0006] On viral diseases, damages from various kinds of viral fishdiseases has begun to be reported since 1980's. For example, flounderrhabdovirus (HRV: 200 nm overall length, bullet form with an envelope)spread over flounder farms in Hokkaido in the first half of 1980's, andit is reported that 80% of flounder fry died then. In addition, it issaid that the amount of damage of salmon family fish by infectioushematopoietic necrosis virus (IHNV: 200 nm overall length, bullet formwith an envelope) is estimated to be 2 to 3 billion yen/year, andseveral 10s billion yen/year in the world. So, many people who involvefish-farming are cautious universally thereon.

[0007] However, concerning viral disease countermeasure for thecultivation fish, diagnostic agents and prevention vaccine are beinginvestigated, but the development has not yet advanced so much. Forexample, vaccine for the iridovirus which was very often reported incultivating red sea bream and so on was developed in 1999. However,since the vaccine must be injected into each fish, it is not widely usedin farms.

[0008] On the other hand, sea lettuce is a kind of green alga growing inseawater, and it occurs in mass by the eutrophication which containsmuch nitrogen and phosphorus in beach and a seawater lake. Therefore,the existence of sea lettuce has been regarded as one of indications toshow the eutrophication state of a sea area conventionally. It isassumed that self-purification action by sea lettuce is very importantsince sea lettuce takes in nitrogen and phosphorus for proliteration. Onthe other hand, the present situation is that people rack their brainsabout disposal of sea lettuce, and its effective utilization is desired.From this point of view, sea lettuce is a practically safe food sourcewhich is partly utilized as food, and the basic study aiming atapplication to feed additives for domestic animal is started, payingattention on nutrition components such as proteins, vitamins, minerals,and so on. However, decisive effective utilization of sea lettuce hasnot been found yet, and development of the effective usage that isaiming at mass consumption is expected.

[0009] The present inventors have investigated in the past for makinggood use of sea lettuce. As a result, the present inventors have foundthat D-cysteinolic acid, which is a sulfur-containing amino acid in sealettuce, reduces internal neutral fat and inhibits the Fentton reactionwhich generates harmful radicals, and filed already patent applicationsbased on the observations. (Jpn. Pat. Appln. Publication Nos.2000-202404 and 2000-103724).

[0010] However, in terms of effective utilization of sea lettuce, moresufficient analysis and assessment are expected at various points ofview about components of sea lettuce.

[0011] In view of the circumstances mentioned above, the presentinvention has been achieved as part of a study to search bioactivecomponents of sea lettuce and to promote effective use thereof.

BRIEF SUMMARY OF THE INVENTION

[0012] A first object of the present invention is to provide anti-viralagents by use of sea lettuce, namely a material which is already ensuredthe safety as food.

[0013] A second object of the present invention is to provide feedshaving antiviral effect by use of sea lettuce.

[0014] The present inventors eagerly analyzed and examined about variousbioactivity such as antitumor, antibacterial, antimelanin, nerve cellpromotion, and antiviral activities on sterile sea lettuce. As a result,it has been found that sea lettuce has antiviral activity, andtherefore, the present invention has been established. There has been,so far, no report that sea lettuce and its extract have suchphysiological activity that inhibits virus infection.

[0015] Accordingly, the antiviral agents of the present invention ischaracterized by containing sea lettuce or an organic solvent extractionfraction thereof.

[0016] In addition, the feeds of the present invention is characterizedby containing the antiviral agents along with feeding components.

[0017] Additional objects and advantages of the invention will be setforth in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention may be realized and obtained bymeans of the instrumentalities and combinations particularly pointed outhereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0018] The accompanying drawings, which are incorporated in andconstitute a part of the specification, illustrate embodiment of theinvention, and together with the general description given above and thedetailed description of the embodiment given below, serve to explain theprinciples of the invention.

[0019]FIG. 1 is a diagrammatic chart showing a step of extractingantiviral components from sea lettuce and a step of processing;

[0020]FIG. 2 is a diagrammatic chart showing an inhibitory effect of thesea lettuce extract against flounder rhabdovirus;

[0021]FIG. 3 is a diagrammatic chart showing an inhibitory effect of thesea lettuce extract against salmon family fish infectious hematopoieticnecrosis virus; and

[0022]FIG. 4 is a diagrammatic chart showing an inhibitory effect ofvarious sea lettuce extracts against salmon family fish infectioushematopoietic necrosis virus.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The term “sea lettuce” in the present invention means algaebelonging to Ulva genus which exist in nature, floating in inlets or thelike of the sea. Sterile sea lettuce (Ulva lactuca) is preferred as sealettuce to use with the present invention since it can be easilyproduced by cultivation through the year.

[0024] In the present invention, when an antivirally active component isextracted from sea lettuce, sea lettuce itself (raw algae) or driedalgae may be used as raw material. When an antiviral component isextracted from sea lettuce, it is effective to extract from sea lettucewith such amount of organic solvent that is several 10s times by weightper dry weight of the sea lettuce. Further in case of requiring anextract in high purity, high purity extraction can be attained byextracting with several times of water or phosphate buffer (pH 7) byvolume per dry weight to extract aqueous fractions, and then, furtherextracting the resulting residue by adding several 10s times by weightof organic solvent per dry weight of sea lettuce. The organic solventused here includes, but not limited to, methanol, ethanol, chloroformand the like. The organic solvent may be removed from this extract undera reduced pressure or the like, and the extract can be dissolved in 1%dimethyl sulfoxide (DMSO). Furthermore, the product can be dried byvacuum drying or the like, and it can be saved for a long period in theform of powders.

[0025] Organic solvent extraction fraction of the sea lettuce obtainedin this way has effective antiviral activity as shown in examples to bedescribed below. Moreover, the sea lettuce itself also showed effectiveantiviral activity. Therefore, sea lettuce and its organic solventextraction fraction are useful as antiviral agents.

[0026] Furthermore, the present invention provides functional feedshaving antiviral activity, in particular. According to one aspect of theinvention, there is provided a technology to prevent infection of fishdisease viruses by feeding a mixture which is prepared by mixing finepulverized sea lettuce in 1 mm or less, with facilitated exposition of alipophilic fraction, and/or the organic solvent extract with any baitcomposed of known components used conventionally.

[0027] Now, a process for extracting antiviral agents from sea lettuce,and preparing antiviral feeds from the resultant extract will beexplained below. As shown in FIG. 1, this process comprises:

[0028] 1: a step of collecting sea lettuce;

[0029] 2: a step of extracting sea lettuce with water;

[0030] 3: a step of extracting sea lettuce with organic solvent;

[0031] 4: a step of drying the sea lettuce extract; and

[0032] 5: a step of adding the sea lettuce extract to bait.

[0033] In Step 1 of collecting sea lettuce, the sea lettuce existing innature or the sea lettuce produced by cultivation are recovered andcollected with a harvest basket. As an apparatus for this purpose, theapparatus as disclosed in Jpn. Pat. Appln. Publication No. 11-066247 bythe present inventors can be used.

[0034] In Step 2 of extraction from sea lettuce with water, when mud ismixed with sea lettuce, sea lettuce is washed with water, mixed withwater in a quantity of several 10s times by weight per dry weight of sealettuce, crushed by homogenizer and subjected to centrifugal separationor a mesh filtration to obtain the sea lettuce powdery residue.

[0035] In Step 3 of extracting sea lettuce with organic solvent, theextraction is performed by mixing the sea lettuce residue with suchamount of organic solvent that is several 10s times per dry weight ofsea lettuce. Examples of the organic solvent include ethanol, methanol,chloroform and so on, and methanol or ethanol is preferred because ofease on operation.

[0036] In Step 4 of drying the sea lettuce extract, it is dried byvacuum freeze dryer or the like. The drying process can make the extractpowdery, enabling easy long-term storage. Further, drying of the sealettuce extract facilitates mixing it with bait. When methanol orchloroform was used for extraction, the drying step is performed afterthe organic solvent has been eliminated completely under vacuum reducedpressure. However, when less toxic ethanol was used, there is no need ofremoving the ethanol completely under vacuum reduced pressure, and theextract can be stored as a solution in a small volume of ethanol and thesolution can be used in a step of adding the extract to bait asdescribed below.

[0037] In Step 5 of adding the sea lettuce extract to bait, the sealettuce organic solvent extract described above is mixed with formulafeeds and fish mince to be fed as a bait. This step of adding isclassified in two types, namely a large scale type which is carried outat the place adjacent to a series of sea lettuce extraction steps, and asmall scale type which is carried out on the farm spot before thefeeding. Even in any case, the organic solvent extract of sea lettuce isdesirably mixed with bait at the rate of 0.1 to 2% by weight.

[0038] It should be noted that although an organic solvent extractedfraction of sea lettuce is used as an antiviral component in the processof FIG. 1, sea lettuce itself may be used instead. In this case, any oneof the sea lettuce from Step 1 of collecting sea lettuce and Step 2 ofextracting sea lettuce with water may be used. In any case, appropriaterate of sea lettuce itself to a bait to be mixed is 1 to 20% by weight,preferably 3 to 10% by weight.

EXAMPLES

[0039] The present invention will be explained in more detail by way ofthe following examples.

Example 1 Preparation of Sea Lettuce Extract and Antiviral Activity Test

[0040] Sterile sea lettuce (Ulva lactuca) collected at a sea area inYokohama Sea Park was used for this experiment. Note that, a depositionapplication of the sea lettuce used here was filed as sterile sealettuce MHI-ATRC-1 strain in National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, but thedeposition of this strain was rejected because it is a green alga notapplicable to deposition.

[0041] Phosphate buffer (pH 7) was added to 10 g of sea lettuce as dryweight, and the mixture was crushed. Extraction in water was performedto give aqueous solution, and further extraction from the residue withorganic solvent was performed. The extracted component was dried andweighed. As a result, the component extracted with phosphate bufferweighed 480 mg, the component with chloroform weighed 1000 mg, thecomponent with methanol weighed 1440 mg, and the component with ethanolweighed 1520 mg. In case of ethanol and methanol both of which arepreferable because of easy use, almost same weight of crude extract wasobtained. Each extracted fraction was dissolved in 1% dimethylsulfoxide, and this solution was used for assessing antiviral activity.

[0042] Antiviral activity of the sea lettuce extract was assessed usingtwo kinds of fish disease viruses: flounder rhabdovirus (HRV8401-Hstrain/host: EPC cell derived from carp) and infectious hematopoieticnecrosis virus of salmon family fish (IHNVChAb strain/host: CHSE-214cell derived from king salmon embryo). After contacting the sea lettuceextract with virus solution for one hour, a virus was inoculated intovarious host cells. After inoculation, the virus was cultured for 7 dayson dish, and a number of plaques (a group of cells which was dissolvedby means of viral infection appeared in the shape of a pin hole) whichappear by the cultivation was counted. Percentage reduction was measuredby assuming the number of plaques without adding the sea lettuce extractas 100.

[0043] Virus infection inhibitory effect of the methanol extract andphosphate buffer extract of sea lettuce was examined by using the twokinds of viruses. As shown in FIGS. 2 and 3, eventually the methanolextract inhibited infection of two kinds of viruses (IHNV; HRV)depending upon the concentration of the extract. Plaque was completely(100%) inhibited at a concentration of not less than 50 μg/ml of themethanol extract. As shown in FIGS. 2 and 3, the inhibition of theplaque formation was hardly seen in the phosphate buffer extract of sealettuce.

[0044] Furthermore, antiviral activity of the ethanol extract andchloroform extract were compared on IHNV which was inhibited by themethanol extract. As a result, comparable virus inhibitory effect wasobserved in three kinds of organic solvent extracts (methanol, ethanol,and chloroform) as shown in FIG. 4. It can be considered that thecomponent having this antiviral activity be a lipophilic substance.Although the mechanism of action is not clear, but it is consideredthat, because the active component of the organic solvent extract of sealettuce is lipophilic, the active component exhibits the antiviraleffect by acting on the envelope portions of virus (membrane composed offat and protein).

Example 2 Addition of Sea Lettuce to Bait and Antiviral Activity

[0045] Addition of sea lettuce to bait was performed by adding finepowder of dry sea lettuce to the conventional formula feeds mixed withfish oil, vegetable oil, or the like. Addition concentration of solidwas 10% by weight. The addition of the sea lettuce extract was performedby adding fine powder of the extract to the formula feeds mixed withfish oil, vegetable oil or the like. The solid was added with 2%concentration by weight. Both of thus obtained two mixed feeds can keeptheir shape even in water, and a cultured fish (flounder) was able to beeasily ingest.

[0046] In order to confirm antiviral activity of sea lettuce, viraltolerance of the flounder adult fish treated with sea lettuce or itsmethanol extract was examined. After feeding bait containing 10% byweight of fine powder of sea lettuce or 2% by weight of its methanolextract to each group of 100 flounders infected with HRV virus, and theywere bred for 5 weeks, the survival rate thereof was examined. Theresult is shown in Table 1. As shown in Table 1, the survival rate ofthe group treated with normal bait markedly lowered to 59%. On the otherhand, the group treated with sea lettuce powder showed 94% of survivalrate, and the group treated with the methanol extract showed 96% ofsurvival rate. TABLE 1 Survival rate of flounder Feeding conditions 2%of 10% of sea Condition sea lettuce for virus No lettuce extractinoculation addition added added Virus 59% 94% 96% inoculated No virus95% 96% 97% inoculated

[0047] In addition, the survival rate in the blank test in which theflounder without infection of HRV virus was cultured is shown below asfollows: 95% in case of no sea lettuce added; 96% in case of powdery sealettuce added; and 97% in case of methanol extract of sea lettuce added.Thus, this had no influence on the antiviral activity described above.

[0048] From the result described above, resistance against viral diseasecan be conferred to adult fish, namely reduction of the death rate byviral diseases can be achieved by feeding sea lettuce or an organicsolvent extract thereof which contains antiviral components.

[0049] Additional advantages and modifications will readily occur tothose skilled in the art. Therefore, the invention in its broaderaspects is not limited to the specific details and representativeembodiments shown and described herein. Accordingly, variousmodifications may be made without departing from the spirit or scope ofthe general inventive concept as defined by the appended claims andtheir equivalents.

What is claimed is:
 1. Antiviral agents comprising sea lettuce or anorganic solvent extraction fraction thereof as an active component. 2.Use of the antiviral agents according to claim 1, wherein the agent areused for therapy or prophylaxis of fish diseases.
 3. Antiviral feedscomprising the antiviral agents according to claim 1 along with feedsfor animal.
 4. A process for preparing antiviral agents comprising: astep of extracting sea lettuce with water for removing water-solublecomponents from the collected sea lettuce; a step of extracting alipophilic fraction from the residue obtained by the step of extractingwith water; and a step of drying the lipophylic fraction obtained fromthe step of extracting with organic solvent.